Process for preparing antibiotic L 17392 (deglucoteicoplanin)

ABSTRACT

Process for preparing antibiotic L, 17392 by catalytically hydrogenating a deglucoteicoplanin ester of formula ##STR1## wherein A, B and Z represent hydrogen atoms, R represents benzyl or substituted benzyl, wherein the phenyl group is substituted with at least a substituent selected from chloro, bromo, fluoro, nitro, (C 1  -C 3 )alkyl, (C 1  -C 3 )alkoxy and the like, with the exclusion of the tri-nitro phenyl group, or acid addition salts thereof, to catalytic hydrogenolysis in the presence of a poisoned hydrogenation catalyst at a temperature from 10° C. to 40° C. and a pressure between ambient pressure and 5 atm, in an inert organic solvent preferably in the presence of a mineral acid.

Antibiotic L 17392 is an antibiotic substance which is obtained byremoving all the sugar moieties from the glycopeptidic antibioticteicoplanin. Teicoplanin is the international non-proprietary name (INN)of the antibiotic substance formerly named teichomycin which is obtainedby cultivating the strain Actinoplanes teichomyceticus nov. sp. ATCC31121 in a culture medium containing assimilable sources of carbon,nitrogen and inorganic salts (see U.S. Pat. No. 4,239,751). According tothe procedure described in the above cited patent an antibiotic complexcontaining teichomycin A₁, A₂ and A₃ is recovered from the separatedfermentation broth by extraction with a suitable water insoluble organicsolvent and precipitation from the extracting solvent according tocommon procedures. Teichomycin A₂, which is the major factor of theisolated antibiotic complex, is then separated from the obtainedantibiotic mixture by means of column chromatography on Sephadex®.

PHYSICO-CHEMICAL CHARACTERISTICS OF CRYSTALLINE PURE ANTIBIOTIC L 17392

(a) it is soluble in water at a pH higher than 9 and aqueous methanol,ethanol and acetone; slightly soluble in ethyl alcohol anddimethylformamide

(b) an ultraviolet absorption spectrum which shows the followingabsorption maxima:

in 0.1N hydrochloric acid: λ_(max) 279 nm (E₁ cm^(1%) =87.1)

in 0.1N sodium hydroxide: λ_(max) 297 nm (E₁ cm^(1%) =165.3)

(c) an infrared absorption spectrum in nujol with the following mainlysignificant absorption maxima (cm⁻¹):

3250 (νNH; and phenolic νOH)

1645 (Amide I)

1610 (νCOO⁻)

1595 (δNH₃ ⁺)

1520 (Amide II)

(d) Some of the ¹ H NMR data obtained after D₂ O exchange and selectivedecoupling experiments of the ¹ H NMR spectrum registered at 270 MHzwith a Bruker WH-270 Spectrometer, in DMSO-d₆ at 50° C. (internalstandard TMS, δ=0.00 ppm) are as follows: (δ, multiplicity):

2.85-3.30, 2 dd; 4.12, dd; 4.37, d; 4.45, d; 4.50, s; 5.00, ddd; 5.11,d; 5.14, d; 5.35, d; 5.56, d; 5.60, d; 6.3-7.9, m; 6.55, d; 7.37, d;7.50, d; 7.61, d; 8.26, d; 8.28, d; 8.5-10.2, br;

d=doublet

dd=doublets of doublets

ddd=doublet of doublets of doublets

s=singlet

m=multiplet

br=broad

(e) an elemental analysis which indicates the following approximatepercentage composition (average): carbon 58.05%; hydrogen 3.58%;nitrogen 8.23%; chlorine 5.85%; (after correction for a weight loss of11%, measured by thermal gravimetric analysis)

(f) a molecular weight of 1199, confirmed also by FAB-MS analysis

(g) the following formula [calculated on the basis of the availabledata]:

    C.sub.58 H.sub.45 Cl.sub.2 N.sub.7 O.sub.18

(h) a retention time (t_(R)) of 12.2 min when analyzed by HPLC using apre-column (5 cm) packed with Perisorb RP-8 (30 μm; Merck) followed by acolumn Hibar RT 250-4 (Merck) prepacked with LiChrosorb RP-8 (10 μm) andeluting with a liner step-gradient ranging from 10% to 30% acetonitrilein 0.2% aqueous ammonium formate; flow rate: 2 ml/min. (internalstandard: Teicoplanin A₂ component 2 of U.K. Patent ApplicationPublication 2121401, t_(R) =22.4 min)

(i) an acidic function capable of forming salts

(l) a basic function capable of forming salts

(m) no sugar residue

On the basis of the chemico-physical data the following formula may beattributed to antibiotic L 17392: ##STR2## A substance having the samestructural formula is disclosed in European Patent Application No.0098578 and is named antibiotic A 41030 factor B. This substance isobtained by mans of a microbiological process which involves thefermentation of the strain Streptomyces virginiae NRRL 12525 orStreptomyces virginiae NRRL 15156 in a suitable medium, the isolation,purification and separation into its components of antibiotic A 41030factor B, included.

Antibiotic L 17392 possesses acid and basic functions capable of formingsalts with bases and acids, respectively. The acid and/or basic salts ofantibiotic L 17392 can be in general prepared according to known per seprocedures.

These procedures includes reacting antibiotic L 17392 with at least amolar equivalent of the selected acid or base.

A preferred procedure for preparing a salt of antibiotic L 17392 with abase includes reacting antibiotic L 17392 and the base in aboutequimolecular amount in water, conveniently at room temperature, andlyophilizing the solution obtained at the end of the salificationreaction.

A preferred procedure for preparing a salt of antibiotic L 17392 with anacid includes reacting antibiotic L 17392 with the selected acid in anaqueous lower alkanol, conveniently at room temperature. Then, the wateris eliminated and the desired salt of antibiotic L 17392 is precipitatedby adding a non-solvent, such as ethyl ether, to the organic phase. Apreferred example of aqueous lower alkanol is a mixture of water andbutanol. The preferred ratio water/butanol is about 30:70.

Representative examples of salts of antibiotic L 17392 with bases arethe alkali metal, such as sodium or potassium, the ammonium andalkylammonium salts. These salts with bases encompass also the saltswith basic amino acids such as lysine and arginine.

Representative examples of salts of antibiotic L 17392 with acids arethe hydrochloric, hydrobromic, sulfuric, phosphoric, glycolic, lactic,pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic,maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,2-phenoxybenzoic, methanesulfonic and 2-hydroxyethanesulfonic acidadditions salts.

The in vitro antibacterial activity of L 17392 was determined using thetwo-fold dilution method in microtiter system. Todd-Hewitt broth (Difco)was used for streptococci and Isosensitest broth (Oxoid) forstaphylococci and gram-negative bacteria. Overnight broth cultures werediluted so that the final inoculum was about 10⁴ colony-forming unitsper ml (cfu/ml). Minimal inhibitory concentration (MIC) was read as thelowest concentration which showed no visible growth after 18-24 hincubation at 37° C. The obtained results are reported in Table I below:

                  TABLE I                                                         ______________________________________                                        In vitro antibacterial activity of L 17392                                                        MIC (μg/ml)                                            Organism            antibiotic L 17392                                        ______________________________________                                        Staphylococcus aureus ATCC 6538                                                                   0.025                                                     Staplylococcus aureus Tour                                                                        0.05                                                      Staphylococcus aureus Tour.sup.(a)                                                                0.2                                                       Staphylococcus aureus Tour.sup.(b)                                                                0.2                                                       Staphylococcus epieermidis ATCC 12228                                                             0.0125                                                    Streptococcus pyogenes C 203                                                                      0.05                                                      Streptococcus pneumoniae UC 41                                                                    0.05                                                      Streptococcus faecalis ATCC 7080                                                                  0.1                                                       Escherichia coli SKF 12140                                                                        25                                                        Proteus vulgaris × 19 H ATCC 881                                                            50                                                        Pseudomonas aeruginosa ATCC 10145                                                                 >100                                                      ______________________________________                                         .sup.(a) Inoculum: 10.sup.6 cfu/ml                                            .sup.(b) Determined in the presence of 30% bovine serum                  

Antibiotic L 17392 was found to be very active against staphylocci (S.aureus, S. epidermidis). In particular, it was very effective againstvarious clinical isolate methicillin-resistant staphylococci (S. aureus,S. epidermidis). Some experimental results are reported in Table II:

                  TABLE II                                                        ______________________________________                                        Organism        MIC (μg/ml)                                                ______________________________________                                        S. aureus L 1096                                                                              0.05                                                          S. aureus L 1097                                                                              0.05                                                          S. aureus L 1524                                                                              0.1                                                           S. aureus L 1526                                                                              0.05                                                          S. epidermidis L 785                                                                          0.05                                                          S. epidermidis L 835                                                                          0.05                                                          S. epidermidis L 1142                                                                         0.05                                                          S. epidermidis L 1372                                                                         0.2                                                           S. epidermidis L 1378                                                                         0.05                                                          ______________________________________                                    

It is known that the removal of all sugars moieties from a complicatedmolecule such as a glycopeptidic antibiotic substance always has manydifficulties. In fact, mild acid conditions usually produce only apartial removal of the sugar moieties while stronger acid hydrolysisconditions may promote partial degradation of the substrate and/orchanges in the stereochemical configuration of the chiral centers. Forinstance, the true aglicone of the glycopeptide antibiotic namedavoparcin has never been isolated, since for this substance, as withsimilar substances, it has not yet been possible to devise selectivehydrolysis conditions capable of removing all the sugar moieties withoutaltering the "core peptide" structure. The following scientificliterature supports the above considerations: G. A. Ellestad et al., J.Antibiotics, 36, 1683 (1983); C. M. Harris et al., J. Am. Chem. Soc.,105, 6915 (1983); W. J. McGahren et al., J. Antibiotics, 36, 1671(1983).

There is no indication whatsoever in the above cited literature whichsuggests using any specific hydrolysis condition to transform ateicoplanin compound or teicoplanin-like compound into the correspondingteicoplanin aglycone (antibiotic L 17392 or deglucoteicoplanin).Moreover, the removal of all the sugar moieties from a teicoplanin-likecompound (as herein below defined) to give antibiotic L 17392 must takeplace without simultaneously provoking any modification or alteration ofthe chemical structure or chiral centers of the substrate, since thesemodifications affect the biological activity of the resulting substanceunfavorably.

The process of the present invention is directed to the production ofantibiotic L 17392 by submitting a suitable deglucoteicoplanin ester tocatalytic hydrogenolysis.

This deglucoteicoplanin ester may be one of the ester derivatives at thecarboxy function of the aglycone moiety of teicoplanin characterized byhaving the ester bond which is cleavable by means of catalytichydrogenation.

Representative examples of deglucoteicoplanin esters which are suitablestarting materials in the process of the invention are: benzyl,substituted benzyl, benzhydryl, 4-picolyl esters and the like.

The term "substituted benzyl" indicates a phenylmethyl group which issubstituted at the phenyl ring with at least one substituent, andpreferably from 1 to 3 substituents, selected from chloro, bromo,fluoro, nitro, (C₁ -C₃)alkyl, (C₁ -C₃)alkoxy and the like, with theexclusion of the tri-nitro phenyl group. Example of said substitutedbenzyl groups are: 3-chlorobenzyl, 4-chlorobenzyl, 2,3-dichlorobenzyl,2,4-dichlorobenzyl, 2,4,6-trichlorobenzyl, 3-fluorobenzyl,4-fluorobenzyl, 3-methylbenzyl, 3-methoxybenzyl, 2-ethoxybenzyl, and thelike.

The catalyst employed in the hydrogenolysis of the invention may be oneof a number of known catalysts, such as Palladium, nickel, Copper,Cobalt either at the zero-valent state and/or with positive oxidationnumbers on suitable supports as known in the art, provided that they areused as "poisoned" catalysts.

A preferred metal catalysts is Palladium on a support selected fromcarbon, barium carbonate, barium sulfate, and calcium sulfate. Goodresults in the process of the invention are obtained by using 5-10%Palladium on barium sulfate. This catalyst is the most preferred one.The reaction is generally conducted in an inert organic solvent, i.e. anorganic solvent which does not unfavorably interfere with the reactioncourse.

Representative examples of suitable inert organic solvents are loweralkanols which are liquid at the reaction temperature and preferablythose which are liquid at room temperature such as methanol and ethanol,dioxane, glycols and glycol monoalkyl ethers, such as ethylene glycoland ethylene glycol monomethyl ether.

Generally, the reaction is conducted in an acidic medium.

Preferred acids to be added to the eaction mixture are rather strongmineral acids, such as hydrohalidic acid, e.g. hydrochloric acid.

The pressure of the reaction medium is, in general, a critical parameterand depends mainly on the type of catalyst used. Generally, it may bebetween ambient pressure and about 5 atm.

The reaction temperature depends on the selected catalyst and pressure.The preferred temperature is room temperature, however, a temperaturefrom 10° C. to 40° C. can be employed, if necessary.

When the catalyst is 5-10% Palladium on barium sulfate in the presenceof mineral acid, the process of the invention is advantageously carriedout at room temperature and pressure. In this case, in fact, it is notnecessary either to increase the temperature or the pressure since thereaction is completed in a reasonable period of time (0.5-3 h) and withvery high yields (80-90%).

When the process of the invention is conducted on an essentially puredeglucoteicoplanin ester or acid-addition salt thereof essentially pureantibiotic L 17392 is obtained.

The reaction course can be easily monitored as known in the art by meansof TLC or HPLC procedures using for instance UV or autobiographicdetection. UV detection is made at about 254 nm, while authobiographicdetection is made using microorganisms which are susceptible to theteicoplanin antibiotics.

The theoretical amount of hydrogen used in the process of the inventionis about 1 mole per mole of ester substrate. Generally, as known in theart, a slight excess of hydrogen is necessary to complete the reaction.

The reaction product is then recovered and purified by means of knownper se techniques such as precipitation by non-solvents, extraction withsolvents, crystallization from solvents and chromatographic procedures,such as column chromatography and reverse-phase column chromatography.

As already said, when a rather purified starting material is used, theresulting antibiotic L 17392 has an acceptable purity. If a furtherpurification of antibiotic L 17392 is necessary or desired, it can beobtained according to usual purification techniques and, in particular,by chromatography, such as "reverse-phase" high performance liquidchromatography (HPLC) and column chromatography.

A preferred purification procedure involves the use of a reverse phasecolumn chromatography. The preferred adsorbent in this case is asilanized silica gel having a distribution particle range from 0.06 to0.2 mm. The eluent can be one of the hydrophilic mixtures that are usedin this purification technique. Representative examples of thesehydrophilic eluents are the mixtures of diluted aqueous solution ofammonium salts of organic acids, acetonitrile or water soluble loweralkanols. Representative examples of diluted aqueous solutions ofammonium salts of organic acids are a 0.1-6% ammonium formate aqueoussolutions, while examples of suitable alkanols are methanol, ethanol,propanol and the like. Preferred eluents are mixtures of aqueousammonium formate and acetonitrile at a pH between 6 and 8 or a mixturesof aqueous ammonium formate and methanol. A preferred procedure includesa first reverse phase chromatography on silanized silica gel (0.06-0.2mm) developing with a linear step gradient of 5 to 21% acetonitrile in0.2% aqueous ammonium formate and a second column chromatography whichuses a mixture of acetonitrile/water 1:1 as the eluent.

Another preferred procedure includes:

(a) mixing a solution of the crude antibiotic in 0.2% aqueous ammoniumformate/methanol/n-butanol, 1:2:3, with silanized silica gel andstripping off the solvents,

(b) applying the residue at the top of a silanized silica gel (0.06-0.2mm) column, developing with 0.6% aqueous ammonium formate andacetonitrile, 9:1, discarding the eluate and continuing the elution witha linear gradient of acetonitrile in water from 1:9 to 4:6.

An example of the way in which the course of the reaction process of theinvention may be monitored or the reaction product titrated by HPLC isas follows: samples are drawn from the reaction mixture at predeterminedtimes, diluted to a final concentration of about 2 mg/ml in a mixture0.2% ammonium formate/acetonitrile, 50:50 (v/v) and injected (20 μl)into the HPLC system. The HPLC system is a chromatograph Varian 5000equipped with 20 μl loop injector Rheodyne 7125; a UV detector at 254 nmand a pre-column packed with Perisorb RP-8 Merck (30-40 μm) followed bya Hibar Merck column (25 cm) pre-packed with LiChrosorb RP-8 (10 μm)Eluent: a linear gradient from 5% B in A to 60% B in A in 30 min, at aflow rate of about 3 ml/min; solution A: 0.2% aqueous ammonium formate;solution B: acetonitrile.

The product obtained according to the above procedure is essentiallypure antibiotic L 17392 which possess satisfactory physico-chemical andbiological characteristics for the use according to the presentdescription. Antibiotic L 17392 can also be obtained as a crystallinepure substance either directly from a crude reaction product or bytreatment of substantially pure antibiotic L 17392.

Crystalline pure antibiotic L 17392 is in fact obtainable by suspendingthe substantially pure amorphous antibiotic in a mixture of water andacetonitrile, 9:1 at a pH of about 1.7. The pH is conveniently adjustedby adding 1N hydrochloric acid. The resulting solution is purified bycolumn chromatography and then the pooled antibiotic L 17392containging-fractions are left aside about 24 h to permit theprecipitation of the crystalline antibiotic L 17392.

A preferred column chromatography procedure in this case comprisesapplying the acidic solution to a silanized silica gel columnequilibrated with diluted aqueous ammonium formate, washing with waterand developing with a linear gradient of acetonitrile in water from 10%to 40%; flow rate 70 ml/h, for 30 h.

This procedure can also be applied directly to crude antibiotic L 17392or to a solution of crude antibiotic L 17392 as obtained after remotionof the catalyst at the end of the hydrogenolysis process hereindescribed. Antibiotic L 17392 crystals are colorless needles.

The term "essentially pure" as referred to an antibiotic substance ofthe present disclosure refers to substances having an HPLC titer greaterthan 95% (percent of the areas of the peaks, at the pre-determined UVwavelength, generally 254 nm), a water and solvents content from 10 to15% (by weight) and an inorganic residue lower than 0.5% (by weight).The deglucoteicoplanin ester derivatives which are the startingmaterials of the process of the invention are represented by thefollowing formula II ##STR3## wherein R is the alkyl residue of analcohol which forms an ester bond with the adjacent carboxy group whichis cleavable by means of catalytic hydrogenolysis, as above defined, andA, B and Z represent hydrogen atoms. These deglucoteicoplanin esters areprepared by submitting a suitable teicoplanin-like substance to anesterification process under controlled conditions. For convenienceherein, the term "teicoplanin-like" substance or substrate represents acompound selected from teicoplanin, a teicoplanin factor, antibiotic L17054, antibiotic L 17046, and mixtures thereof in any proportion.

Some of these teicoplanin-like substances are represented by the aboveformula II wherein R and R¹ represent hydrogen, A is hydrogen or N-[(C₁₀-C₁₁)aliphatic acyl]-β-D-glucosamine, B is hydrogen, or aN-acetyl-β-D-glucosamine, and Z is hydrogen or α-D-mannose. As alreadysaid, teicoplanin is an antibiotic substance obtained by Actinoplanesteichomyceticus ATCC 31121 and is disclosed in U.S. Pat. No. 4,239,751.

British Patent Application Publication No. 2121401 discloses thatantibiotic teicoplanin (formerly teichomycin) factor A₂ is a mixture offive closely related co-produced components. According to recentstructural studies it is possible to represent teicoplanin A₂ components1, 2, 3, 4 and 5 by the above formula I wherein R and R¹ are bothhydrogen, A is N-[(C₁₀ -C₁₁)aliphatic acyl]-β-D-glucosamine group, B isa N-acetyl-β-D-glucosamine group and Z is a α-D-mannose group.

All these sugar moieties, when present, are linked to the teicoplaninnucleus through O-glycosidic bonds. Representative and preferredexamples of (C₁₀ -C₁₁)aliphatic acyl groups are n-decanoyl,8-methylnonanoyl, Z-4-decenoyl, 8-methyldecanoyl, and 9-methyldecanoyl.

Antibiotic L 17054 and antibiotic L 17046 are teicoplanin hydrolysisproducts. They are described in European Patent Application Nos.84102666.9 and 84102665.1, respectively. They are obtained by submittingteicoplanin, a pure factor thereof or a mixture of any of said factorsin any proportion, to selective hydrolysis which results in removing oneor two sugar moieties of the starting material.

More particularly, the selective removal of N-[(C₁₀ -C₁₁)aliphaticacyl]-β-D-glycosamine gives antibiotic L 17054, while the selectiveremoval of N-[(C₁₀ -C₁₁)aliphatic acyl]-β-D-glucosamine and α-D-mannosegroups gives antibiotic L 17046.

Preferred hydrolysis conditions for the production of antibiotic L 17054are: about 0.5N hydrochloric acid at a temperature between 70° and 90°C. and for a time which is generally between 15 and 90 min.

Antibiotic L 17054 is represented by the above formula II wherein R andR¹ are hydrogen atoms, A is hydroxy, B is N-acetyl-β-D-glycosamine and Zis α-D-mannose.

Preferred hydrolysis conditions for the preparation of antibiotic L17046 are: about 1-3N hydrochloric acid, at a temperature between 50°and 90° C. and for a time which is generally between 30 and 60 min.

Antibiotic L 17046 is represented by the above formula II wherein R andR¹ are hydrogen atoms, A and Z are hydroxy groups and B isN-acetyl-β-D-glucosamine.

As already said, the deglucoteicoplanin esters of formula II areprepared by submitting a suitable teicoplanin-like substance toesterification under controlled conditions.

The reaction conditions of the esterification procedure are such thatthe "teicoplanin nucleus" is not modified and that all the sugarmoieties of the starting material are hydrolyzed before theesterification is completed. A convenient procedure for preparing thedeglucoteicoplanin ester intermediates of the invention includesreacting a teicoplanin-like compound with an excess of the suitablealcohol of formula ROH, wherein R is as above, in the presence of anacid catalyst such as 37% hydrochloric acid. Preferably, the alcohol offormula ROH is a liquid at the reaction temperature, so that it may actalso as the reaction medium, without the need of adding another suitablesolvent. The reaction is preferably conducted under reduced pressure.The reaction temperature is generally between 50° and 80° C., when thereaction pressure is about 20 mmHg. When necessary, portions of amixture of the 37% hydrochloric acid with the suitable alcohol are addedfrom time to time to reintegrate the portions of reaction medium whichevaporates.

Portions of a suitable inert solvent capable of forming minimumazeotropic mixtures with water are also added and then the azeotropewhich forms is distilled off under vacuum. Representative examples ofsolvents capable of forming minimum azeotropic mixtures with water are:benzene, toluene, butyl, ether, carbon tetrachloride, chloroform,cyclohexane, 2,5-dimethylfurane, hexane, nonane, m-xilene and the like.

These alternated operations of adding alcoholic hydrochloric acidfollowed by the minimum azeotrope-forming solvent and distilling theaqueous azeotrope which forms are repeated several times until thereaction is completed (i.e. the desired ester derivative is produced inacceptable or optimal yields).

PHYSICO-CHEMICAL CHARACTERISTICS OF DEGLUCOTEICOPLANIN BENZYL ESTER,HYDROCHLORIDE

(a) IR registered in a nujol mull with a Perkin-Elmer 850 instrument: νCO ester=1730 cm⁻¹ ;

(b) a relative retention time ##EQU1## in the HPLC system reported abovewhich is 1.69 (t_(R) deglucoteicoplanin 11.4 min.).

(c) a pKa value of a sample dissolved in methylcellosolve®/water 4:1which is 6.67

(d) UV absorption maxima (nm): 280 (in methanol); 279 (in 0.1Nhydrochloric acid); 279 (in phosphate buffer pH 7.4); 298 (in 0.1Nsodium hydroxide);

(e) elemental analysis (determined on samples previously dried at 140°C. under nitrogen atmosphere):

Found: C% 58.57; H% 4.14; N% 7.28; Cl% (total).sup.(b) 7.90; Cl%(ionic).sup.(b) 2.66; inorganic residue %.sup.(c) 0.2; weight loss%.sup.(d) 10.1.

Calculated for C₆₅ H₅₁ Cl₂ N₇ O₁₈ : C% 58.90; H% 3.95; N% 7.40; Cl%(total).sup.(b) 8.02; Cl% (ionic).sup.(b) 2.67.

PHYSICO-CHEMICAL CHARACTERISTICS OF ANTIBIOTIC L 17046

Antibiotic L 17046 has the following characteristics:

(a) the specific rotation [α]_(D) ²⁰ is -44°(c=1%, DMF)

(b) it is freely soluble in water at pH>8.0, in dimethylformamide,dimethylsulfoxide, propyleneglycol and methylcellosolve; slightlysoluble in methanol; almost insoluble in n-hexane, ethyl ether andacetone.

(c) it has an ultraviolet absorption spectrum that exhibits thefollowing absorption maxima:

in 0.1N hydrochloric acid: λ_(max) 278 nm (E₁ cm^(1%) =67.1)

in 0.1N sodium hydroxide: λ_(max) 297 nm (E₁ cm^(1%) =124.1)

in phosphate buffer pH 7.4: λ_(max) 277 nm (E₁ cm^(1%) =75.0)

(d) an infrared absorption spectrum in nujol with the followingobservable absorption maxima (cm⁻¹): 3700-2000, 2970-2850 (nujol), 1655,1610, 1595, 1515, 1490, 1460 (nujol), 1375 (nujol), 1300, 1230, 1145,1060, 1010, 890, 850, 820, 720 (nujol)

(e) an elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere (weight loss=8.4%) , whichindicates the following approximate percentage composition (average):carbon 56.74%; hydrogen, 4.27%; nitrogen, 7.99%; chlorine, 5.11%; ashes,0.6%.

(f) the following R_(f) values in the TLC systems indicated below:

    ______________________________________                                        Elution system (v/v)     R.sub.f value                                        ______________________________________                                        (I) Acetonitrile/water 75:25                                                                           0.53                                                 (silica gel Merck 60 F.sub.254)                                               (II) Acetonitrile/5% aqueous sodium sul-                                                               0.54                                                 fate 30:70                                                                    (silica gel Merck silanized 60 F.sub.254)                                     Visualization: UV-light at 254 nm; 3% ethanolic                               ninhydrine; 1% methanolic fluorescamine;                                      ______________________________________                                    

(g) a retention time (t_(R)) of 10.8 minutes when analyzed by reversedphase HPLC using a 150×4.0 mm Zorbax®ODS (5-6 μm) column (Zorbax is atrademark of the Dupont Co. for a octadecylsilane silica matrix), andeluting with a linear gradient from 0% to 50% solution B in solution Ain 40 minutes

solution A: 25 mM NaH₂ PO₄ /acetonitrile (9:1) buffered at pH 6.0 with0.1N NaOH

solution B: 25 mM NaH₂ PO₄ /acetonitrile (3:7) buffered at pH 6.0 with0.1N NaOH), with a flow rate of 2 ml/min.; (internal standard:3,5-dihydroxytoluene t_(R) 5.60 minutes)

(h) Some of the ¹ H NMR data obtained after D₂ O exchange and selectivedecoupling experiments are as follows (the ¹ H NMR spectrum isregistered at 270 MHz in DMSO-d₆ at 60° C. and with a sampleconcentration of 20 mg/ml; internal standard, TMS δ=0.00 ppm): (δ ppm,multiplicity): 1.86, s; 2.81, d; 3.5, dd; ˜3-4; 4.12, d; 4.32, d; 4.37,d; 4.56, s; 4.95, ddd; 5.07, s; 5.31, d; 5.39, s; 5.51, s; 5.66, d;6.12, d; 6.29, s; 6.32, s; 6.37, s; 6.42, s; 6.60, d; 6.62, s; 6.64, d;6.92, d; 7.90, s; 7.12, d; 7.21, d; 7.25, d; 7.43, d; 7.64, d; 7.66, d;7.70, d; 7.85, s; 8.12, d; 8.46, d; ˜9.5, s.

(i) a potentiometric titration profile which shows three titrationslopes with pH1/2 values equal to 5.0 (one equivalent), 7.0 (oneequivalent), and 11 (five equivalents) in methylcellosolve:water 4:1upon titration with 0.01N NaOH of the solution of the test compoundcontaining an excess of 0.01N in the same solvent mixture

(l) an acidic function capable of forming salts

(m) a basic function capable of forming salt

(n) a sugar residue which is N-acetyl-β-D-glucosamine.

PHYSICO-CHEMICAL CHARACTERISTICS OF ANTIBIOTIC L 17054

Antibiotic L 17054 has the following characteristics:

(a) the specific rotation [α]_(D) ²⁰ is -34°(c=1%, DMF)

(b) it is freely soluble in water at pH>8.0, in dimethylformamide,dimethylsulfoxide, propylene glycol and methylcellosolve; slightlysoluble in methanol; almost insoluble in ethyl ether and acetone.

(c) an ultraviolet absorption spectrum which has the followingabsorption maxima:

in 0.1N hydrochloric acid: λ_(max) 278 nm (E₁ cm^(1%) =60.6)

in 0.1N sodium hydroxide: λ_(max) 297 nm (E₁ cm^(1%) =118.8)

in phosphate buffer pH 7.4: λ_(max) 277 nm (E₁ cm^(1%) =70.3)

(d) an infrared absorption spectrum in nujol with the followingdescription maxima (cm⁻¹): 3700-2000, 2970-2850 (nujol), 1655, 1610,1995, 1515, 1490, 1460 (nujol), 1375 (nujol), 1300, 1230, 1145, 1060,1020, 970, 890, 850, 820, 720 (nujol)

(e) an elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere (weight loss=7.8%), which indicatedthe following approximate percentage composition (average): carbon55.46%; hydrogen, 4.50%; nitrogen 7.20%; chlorine 4.67%; ashes 0.2%.

(f) it has the following R_(f) values in the TLC systems indicatedbelow:

    ______________________________________                                        Elution system (v/v)     R.sub.f value                                        ______________________________________                                        (I) Acetonitrile/water 75:25                                                                           0.32                                                 (silica gel Merck 60 F.sub.254)                                               (II) Acetonitrile/5% aqueous sodium sul-                                                               0.61                                                 fate 30:70                                                                    (silica gel Merck silanized 60 F.sub.254)                                     Visualization: UV-light at 254 nm; 3% ethanolic                               ninhydrine; 1% methanolic fluorescamine;                                      ______________________________________                                    

(g) a retention time (t_(R)) of 8.3 minutes when analyzed by HPLC usinga 150×4.0 mm Zorbax® ODS (5-6 μm) column (Zorbax is a trademark of theDupont Co. for an octadecylsilane silica gel matrix), and eluting with alinear gradient from 0% to 50% solution B in solution A in 40 minutes(solution A: 25 mM NaH₂ PO₄ /acetonitrile (9:1) buffered at pH 6.0 with0.1 N NaOH; solution B: 25 mM NaH₂ PO₄ /acetonitrile (3:7) buffered atpH 6.0 with 0.1N NaOH), with a flow rate of 2 ml/min; (internalstandard: 3,5-dihydroxytoluene t_(R) 5.60 minutes)

(h) Some of the ¹ H NMR data obtained after D₂ O exchange and selectivedecoupling experiments are as follows (the ¹ H NMR spectrum isregistered at 270 MHz in DMSO-d₆ at 60° C. and with a sampleconcentration of 20 mg/ml internal standard, TMS δ=0.00 ppm): (δ ppm,multiplicity): 1.88, s; 2.85, d; ˜3.5, dd; 3-4; 4.20, d; 4.48, d; 4.50,d; 4.62, s; 4.96, ddd; 5.18 d; 5.31, s; 5.35, d; 5.39, s; 5.68, d; 5.71,s; 6.20, d; 6.41, s; 6.51, s; 6.56, s; 6.74, d; 6.77, s; 6.80, s; 6.80,d; 6.98, d; 7.08, s; 7.15, d; 7.21, d; 7.28, d; 7.35, d; 7.50, d; 7.56,d; 7.64, d; 7.73, d; 7.86, s; 8.42, d.

(i) a potentiometric titration profile which shows three titrationslopes with pH1/2 values equal to 5.0 (one equivalent), 7.0 (oneequivalent), and 11 (five equivalents) in methylcellosolve:water 4:1upon titration with 0.01N NaOH of the solution of the test compoundcontaining an excess of 0.01N HCl in the same solvent mixture

(l) an acidic function capable of forming salts

(m) a basic function capable of forming salts

(n) two sugar residues which are α-D-mannose andN-acetyl-β-D-glucosamine.

The following examples illustrate the manner in which the invention canbe practiced, but, as such, should not be constructed as limiting itsoverall scope.

EXAMPLE 1 Preparation of antibiotic L 17392 (deglucoteicoplanin) byhydrogenolysis of deglucoteicoplanin benzyl ester, hydrochloride

A solution of 4.9 g of essentially pure deglucoteicoplanin benzyl esterhydrochloride, in 500 ml of a CH₃ OH/0.1N HCl 7:3 (v/v) solution ishydrogenated at room temperature and pressure in the presence of 3.5 gof 5% Pd/BaSO₄. In 30 min about 145 ml of H₂ are absorbed. Thesuspension is filtered and the catalyst is washed thoroughly with 200 mlof a CH₃ OH/H₂ O 1:1 (v/v) solution, then it is discarded. The filtratesare combined, 600 ml of n-butanol is added and the resulting solution isconcentrated to a small volume. By adding acetone a solid separateswhich is collected, washed with acetone, then with ether, and driedunder vacuum at room temperature overnight yielding 4.0 g of essentiallypure antibiotic L 17392, hydrochloride.

EXAMPLE 2 Purification of crude antibiotic L 17392

By following the above example but using crude deglucoteicoplanin benzylester, crude antibiotic L 17392 is obtained with substantially the samepurity as the starting material.

When crude antibiotic L 17392 is obtained, it can be purified accordingto the following procedure: 5.3 g of crude antibiotic L 17392 (titer60%) is dissolved in 1 liter of a mixture of 0.2% aqueous ammoniumformate/methanol/n-butanol, 1:2:3 (v/v/v) and silanized silica gel(0.06-0.2 mm; Merck 60) (20 g) is added thereto. After appropriatestirring, the solvents are stripped off under vacuum and the residue isapplied to the top of a chromatographic column prepared with 750 g ofsilanized silica gel (0.06-0.2 mm; Merck) in water. The column isdeveloped with 1 l of a mixture of 0.6% aqueous ammonium formate withCH₃ CN, 9:1 (v/v). The eluate is discarded, then the elution iscontinued with a linear gradient of acetonitrile in water from 1:9 to4:6 at a rate of 200 ml/h for about 30 h.

Fractions of 25 ml each are collected and monitored by HPLC. Thedeglucoteicoplanin containing fractions (200 to 250) are pooled andn-butanol is added. After stirring the mixture is concentrated to asmall volume under reduced pressure, ethyl ether is added thereto andthe solid which separates is collected by filtration, washed with ethylether and dried at 40° C. under vacuum, yielding 0.9 g of essentiallypure antibiotic L 17392.

EXAMPLE 3 Preparation of crystalline pure antibiotic L 17392

The pH of a suspension of essentially pure antibiotic L 17392 (0.9 g) in100 ml of water/acetonitrile, 90:10 (v/v) is brought to 1.7 with 1Nhydrochloric acid, at room temperature. The resulting solution isapplied to a silanized silica gel column (200 g, 0.06-0.2 mm, Merck 60),equilibrated with 1% aqueous ammonium formate, at a rate of 20 ml/h.

Water (300 ml) is passed through the column and the eluate is discarded.Then the column is developed with a linear gradient of acetonitrile inwater from 10% to 40% at a rate of 70 ml/hour for 30 hours. Fraction of7 ml each are collected and monitored by HPLC.

Antibiotic L 17392 enriched fractions (221-239) are pooled and allowedto stand for 24 hr at room temperature. The solid precipitate iscollected by filtration, washed with a small amount (10 ml) ofacetonitrile and then with ethyl ether (100 ml) and re-crystallized froma mixture water/acetonitrile 80:20 (v/v). The crystalline solid thusobtained is collected by filtration, washed with ethyl ether and finallydried under vacuum (2 mmHg) for three days yielding 0.55 g ofcrystalline pure antibiotic L 17392 (colorless needles).

EXAMPLE 4 Preparation of antibiotic L 17392 hydrochloride

Crystalline pure antibiotic L 17392 as obtained in the foregoing example(130 mg) is suspended in a mixture (12 ml) acetonitrile/water), 2:3(v/v) and 1N hydrochloric acid (0.2 ml) is added thereto. After addingn-butanol (15 ml), the resulting solution is concentrated to a smallvolume (about 2 ml) under reduced pressure (about 20 mmHg) at 40° C.Ethyl ether (10 ml) is added thereto and the precipitate which forms iscollected by filtration, washed with ethyl ether, and dried underreduced pressure at about 50° C., overnight, yielding 107 mg ofantibiotic L 17392 hydrochloride.

PREPARATION OF THE STARTING MATERIALS Preparation of antibioticdeglucoteicoplanin benzyl ester, hydrochloride

(a) by treatment of antibiotic L 17046 with benzyl alcohol 1M hydrogenchloride

A suspension of 18 g (10 mmol) of essentially pure antibiotic L 17046 in600 ml of 1M hydrogen chloride in benzyl alcohol is stirred at 60° C.After 15 minutes a clear solution forms which is stirred for additional3 h at the same temperature, then the solution is cooled to 15° C. andstirring is continued at room temperature for additional 12 h. By adding4 l of a mixture n-hexane/ether 4:3 (v/v) a solid separates which iscollected, washed with 1 l of ether and re-dissolved in 150 ml ofmethanol. The solution is diluted with 1 l of H₂ O and extracted (pH2.5) twice with 2 l of ethyl acetate. The organic layers are combinedand a mixture of 10 ml of 1N HCl in 200 ml of n-butanol is added, thenthe solution is concentrated in a small volume. By adding 1 l of amixture n-hexane/ether 3:2 ) (v/v) a solid separates which is collected,washed with ether and dried under vacuum at 40° C. for 8 h yielding 8.5g of deglucoteicoplanin benzyl ester, hydrochloride (analysis:deglucoteicoplanin benzyl ester, hydrochloride 70%, water and solvents15%, undefined impurities 15%).

(b) by treatment of the antibiotic L 17054 with 1M hydrogen chloride in90% aqueous benzyl alcohol

To a stirred suspension of 10 g of essentially pure antibiotic L 17054in 90 ml of benzyl alcohol, 10 ml of 37% hydrochloric acid is added at40° C. The reaction mixture is heated to 70° C. and stirring iscontinued for 30 minutes, then water is completely removed under vacuum(about 20 mmHg) at 70° C. (bath temperature). Benzene is added and thenthe mixture is evaporated under reduced pressure to remove any aqueousresidue by means of azeotropic distillation. Then the mixture is dilutedwith 100 ml of 1M hydrogen chloride in aqueous benzyl alcohol (preparedas above). The clear solution so obtained is stirred at 65° C. for 6 h,then cooled to 15° C. and worked up as described in the foregoing point(a), yielding 4.85 g of deglucoteicoplanin benzyl ester, hydrochloride(analysis: deglucoteicoplanin benzyl ester, hydrochloride 75%, water andsolvents 15%, undefined impurities 10%).

(c) by treatment of teicoplanin with 2M hydrochloric acid in 80% aqueousbenzyl alcohol, under vacuum and with repeated additions of benzene and37% hydrochloric acid.

To a stirred suspension of 10 g of teicoplanin (content in teicoplanincomponents: 85%) in 80 ml of benzyl alcohol, 20 ml of 37% hydrochloricacid is added at 40° C. The mixture is kept under vacuum (about 20 mmHg)for about 60 minutes while heating to about 65° C. (bath temperature),then 50 ml of benzene is added and the mixture is evaporated undervacuum at about 65° C. After 30 minutes, a mixture of 5 ml of 37%hydrochloric acid in 25 ml of benzyl alcohol is added to the reactionmixture which is then re-submitted to the "under vacuum" procedure(about 20 mmHg; about 65° C.) for 30 minutes. Then 50 ml of benzene isadded and evaporated as previously described. Alternate additions ofmixtures of 5 ml of 37% hydrochloric acid in 15 ml of benzyl alcohol andof 50 ml of benzene separated by the "under vacuum" procedure arerepeated every 30 minutes for 8 hours. Then 20 ml of 37% hydrochloricacid and 100 ml of benzene are added, while water and benzene areevaporated under vacuum and the resulting clear solution is stirred atroom temperature and pressure under argon atmosphere for 12 h, then thereaction mixture is poured into 1.5 l of ether. A solid separates whichis collected, washed with ether and dried under vacuum at roomtemperature overnight, yielding 10 g of the crude ester of the title.This product is dissolved in 150 ml of methanol and 300 ml of water and300 ml of ethyl acetate are added thereto with vigorous stirring. Afterfew minutes additional 300 ml of water, 300 ml of ethyl acetate and amixture of 300 ml of n-butanol/water 1:2 (v/v) were added. The pH of theaqueous layer is adjusted to 3.5 and the organic phase is separated. Theaqueous phase is extracted twice with ethyl acetate (600 ml each time).The organic layers are combined, washed with 400 ml of water andconcentrated to a small volume under vacuum. By adding ether a solidseparates which is collected, washed with ether and dried under vacuumat room temperature overnight, yielding 6.1 g of crudedeglucoteicoplanin benzyl ester, hydrochloride (analysis:deglucoteicoplanin benzyl ester, hydrochloride 65%; water and solvents15%, undefined impurities 20%).

PURIFICATION OF DEGLUCOTEICOPLANIN BENZYL ESTER BY SILICA-GEL COLUMNCHROMATOGRAPHY

Silica-gel (0.06-0.2 mm, Merck 60) (10 g) is added to a solution of 2.5g of crude deglucoteicoplanin benzyl ester (titer 65%) in 100 ml of 90%aqueous methanol. The solvent is completely evaporated under vacuum andthe residue is applied to a chromatographic column containing 250 g ofsilica-gel slurried in acetonitrile. The column is developed bysequentially using the following solvent mixtures:

    ______________________________________                                        CH.sub.3 CN             250 ml                                                CH.sub.3 CN/H.sub.2 O 97:3 (v/v)                                                                      500 ml                                                CH.sub.3 CN/H.sub.2 O 94:6 (v/v)                                                                      500 ml                                                ______________________________________                                    

The eluates are discarded, then the column is eluted with a lineargradient of acetonitrile in water obtained by mixing 1.5 l each of thesolvent mixtures CH₃ CN/H₂ O 94:6 (v/v) and CH₃ CN/H₂ O 70:30 (v/v) at arate of 200 ml/h. Fractions of 25 ml are collected and assayed by HPLC.Deglucoteicoplanin benzyl ester containing fractions are combined (700ml), n-butanolic 0.05M hydrogen chloride (250 ml) is added thereto, andthe solvents are evaporated up to a final volume of about 30 ml. Byadding either (300 ml) a solid separates which is collected, washed withether and dried under vacuum at 40° C. for 48 h, yielding 1.6 g ofessentially pure deglocoteicoplanin benzyl ester, hydrochloride.

By operating essentially following the procedures of the above examplesand employing the suitable reagents the following starting materials canbe obtained:

deglucoteicoplanin 4-chlorobenzyl ester

deglucoteicoplanin 2,4-chlorobenzyl ester

deglucoteicoplanin 4-nitrobenzyl ester

deglucoteicoplanin 3,4-nitrobenzyl ester

and the acid addition salts thereof.

We claim:
 1. A process for preparing antibiotic L 17392 which has thefollowing characteristics(a) it is soluble in water at a pH higher than9 and aqueous methanol, ethanol and acetone; slightly soluble in ethylalcohol and dimethylformamide (b) an ultraviolet absorption maxima whichshows the following absorption maxima:in 0.1N hydrochloric acid: λ_(max)279 nm (E₁ cm¹ %=87.1) in 0.1N sodium hydroxide: λ_(max) 297 nm (E₁ cm¹%=165.3) (c) an infrared absorption spectrum in nujol with the followingmainly significant absorption maxima (cm⁻¹):3250 (ν NH; and phenolic νOH) 1645 (Amide I) 1610 (ν COO⁻) 1595 (δ NH₃ ⁺) 1520 (Amide II) (d) Someof the ¹ H NMR data obtained after D₂ O exchange and selectivedecoupling experiments of the ¹ H NMR spectrum registered at 270 MHzwith a Bruker WH-270 Spectrometer, in DMSO-d₆ at 50° C. (internalstandard TMS, δ=0.00 ppm) are as follows (δ, multiplicity): 2.85-3.30,2dd; 4.12, dd; 4.37, d; 4.45, d; 4.50, s; 5.00, ddd; 5.11, d; 5.14, d;5.35, d; 5.56, d; 5.60, d; 6.3-7.9, m; 6.55, d; 7.37, d; 7.50, d; 7.61,d; 8.26; d; 8.28, d; 8.5-10.2, br;d=doublet dd=doublets of doubletsddd=doublet of doublets of doublets s=singlet m=multiplet br=broad (e)an elemental analysis which indicates the following approximatepercentage composition (average): carbon 58.05%; hydrogen 3.58%;nitrogen 8.23%; chlorine 5.85%; (after correction for a weight loss of11%, measured by thermal gravimetric analysis) (f) a molecular weight of1199 confirmed also by FAB-MS analysis (g) the following formula[calculated on the basis of the available data]:

    C.sub.58 H.sub.45 Cl.sub.2 N.sub.7 O.sub.18

(h) a retention time (t_(R)) of 12.2 min when analyzed by HPLC using apre-column (5 cm) packed with Perisorb RP-8 (30 μm; Merck) followed by acolumn Hibar RT 250-4 (Merck) prepacked with LiChrosorb RP-8 (10 μm) andeluting with a liner step-gradient ranging from 10% to 30% acetonitrilein 0.2% aqueous ammonium formate; flow rate: 2 ml/min. (internalstandard: Teicoplanin A₂ component 2, t_(R) =22.4 min) (i) an acidicfunction capable of forming salts (l) a basic function capable offorming salts (m) no sugar residue (n) the following putative chemicalformula: ##STR4## which comprises submitting a deglucoteicoplanin esterof formula II ##STR5## wherein A, B and Z represent hydrogen atoms, Rrepresents benzyl or substituted benzyl, wherein the phenyl group issubstituted with at least a substituent selected from chloro, bromo,fluoro, nitro, (C₁ -C₃)alkyl and (C₁ -C₃)alkoxy, with the exclusion ofthe tri-nitro phenyl group, R' is H, or acid addition salts thereof, tocatalytic hydrogenolysis in the presence of a poisoned hydrogenationcatalyst at a temperature from 10° C. to 40° C. and a pressure betweenambient pressure and 5 atm, in an inert organic solvent in the presenceof a mineral acid.
 2. A process according to claim 1 wherein thecatalyst is a poisoned catalyst selected from Palladium, nickel, Copper,and Cobalt either at the zero-valent status or with a positive oxidationnumber on a suitable support.
 3. A process according to claim 1 whereinthe poisoned catalyst is Palladium on carbon, barium carbonate orcalcium sulfate.
 4. A process according to claim 1 wherein the poisonedcatalyst is 5-10% Palladium on Barium sulfate.
 5. A process according toclaim 1 wherein the poisoned catalyst is 5% Palladium on Barium sulfate.6. A process according to claim 1 wherein the inert organic solvent isselected from methanol, ethanol, dioxane, ethylene glycol and ethyleneglycol monomethylether.
 7. A process according to claim 1 wherein themineral acid is hydrochloric acid.
 8. A process according to claim 1wherein the temperature is room temperature.
 9. A process according toclaim 1 wherein the pressure is the ambient pressure.
 10. A processaccording to claim 1 wherein the starting material of formula II isdeglucoteicoplanin benzyl ester or an acid addition salt thereof.
 11. Aprocess according to claim 1 which further comprises suspending thesubstantially pure amorphous antibiotic L 17392 product in a mixture ofwater and acetonitrile, 9:1 at a pH of about 1.7, the pH being adjustedby adding 1N hydrochloric acid, purifying the resulting solution bycolumn chromatography, and leaving set aside for about 24 hours thepooled antibiotic L 17392 containing fractions to permit theprecipitation of crystalline antibiotic L 17392.